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United States Patent |
6,113,766
|
Steiner
,   et al.
|
September 5, 2000
|
Device for rehydration and electrophoresis of gel strips and method of
using the same
Abstract
The present invention involves a gel strip carrier module for a gel strip
that reduces the handling of the gel strip and the hands-on-time during
preparation of the gel strip for isoelectric focusing. The gel carrier
module includes a gel strip chamber that serves as both a rehydration and
focusing chamber, and allows the sample to be applied either throughout
the entire gel or in a defined zone. The gel carrier module includes a
pair of electrodes near opposite ends of the chamber that the gel strip
rests on, gel side facing down. The gel carrier module includes a cover
with hold-down blocks or pressure blocks to assure reliable, light contact
between the gel and the electrodes during focusing. A rehydration buffer
is added into the chamber, and the gel strip is gently placed in the
chamber, gel side down, for rehydration. The rehydration buffer may
include the sample, or, in the event that the sample needs to be applied
after rehydration, the gel carrier module includes sample application
wells between the electrodes that the sample can be added to after
rehydration of the gel strip.
Inventors:
|
Steiner; Urs (Sunnyvale, CA);
Islam; Mohammed Rezaul (Sunnyvale, CA);
Hungerman; Eric R. (Danville, CA)
|
Assignee:
|
Hoefer Pharmacia Biotech, Inc. (San Francisco, CA)
|
Appl. No.:
|
095002 |
Filed:
|
June 9, 1998 |
Current U.S. Class: |
204/606; 204/615 |
Intern'l Class: |
G01N 027/26 |
Field of Search: |
204/456,466,467,606,616,615
|
References Cited
U.S. Patent Documents
4999340 | Mar., 1991 | Hoffman et al. | 514/23.
|
5209831 | May., 1993 | MacConnell | 204/299.
|
5399255 | Mar., 1995 | Sarrine | 204/299.
|
Foreign Patent Documents |
0 304 195 A2 | Feb., 1989 | EP.
| |
0 457 526 A2 | Nov., 1991 | EP.
| |
0 631 133 A2 | Dec., 1994 | EP.
| |
91 10 951 U1 | Nov., 1991 | DE.
| |
96/34276 | Oct., 1996 | WO.
| |
Other References
Immobiline.RTM. DryStrip Reswelling Tray: User Maual Amersham Pharmacia
Biotech (1998).
"Sample application by in-gel rehydration improves the resolution of
two-dimensional electrophoresis with immobilized pH gradients in the first
dimension," Electrophoresis 15:1552-1558 (1994).
"Improved and simplified in-gel sample application using reswelling of dry
immobilized pH gradients," Electrophoresis 18:324-327 (1997).
|
Primary Examiner: Warden, Sr.; Robert J.
Assistant Examiner: Noguerola; Abe
Attorney, Agent or Firm: Malia; Victoria M., Ronning, Jr.; Royal N.
Parent Case Text
RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application Ser.
Nos. 60/048,999, filed on Jun. 9, 1997, 60/049,135, filed on Jun. 10,
1997, and 60/059,810, filed on Sep. 24, 1997.
Claims
What is claimed is:
1. A device for rehydrating and for performing electrophoresis on a gel
strip having two ends and a gel face, comprising:
a holder including a gel strip chamber configured to receive the gel strip
and a rehydration buffer medium wherein the holder includes a floor and a
wall, an inner surface of the wall and upper surface of the floor define
the gel strip chamber, the gel strip chamber includes a first end and a
second end; and
first and second electrodes carried by said holder and having internal
electrical contact points carried by the floor of the gel strip chamber
adapted to contact the gel face near the first and second ends of the gel
strip and external electrical contact points adapted to be electrically
connected to a power supply for performing electrophoresis on the gel
strip with the first and second electrodes, further adapted to contact the
face of the gel strip with said strip oriented to face the bottom of the
chamber, and the device further including a member adapted to maintain the
gel strip in contact with the internal electrical contact points during
electrophoresis of the gel strip, and further wherein
at least one member is adapted to maintain the gel strip in contact with
the internal electrical contact points, that at least one member extends
from the cover and includes a bottom surface and a height, whereby when
the chamber is covered, the distance between the floor and the bottom
surface of the at least one member is approximately equal to the thickness
of the gel strip when rehydrated.
2. The device of claim 1, wherein the holder includes a floor and a wall,
an inner surface of the wall and upper surface of the floor define the gel
strip chamber, the gel strip chamber includes a first end and a second
end, the internal electrical contact point of the first electrode is
carried by the cover and configured to be adjacent to the first end of the
gel strip chamber when the chamber is covered, the internal electrical
contact point of the second electrode is carried by the cover and
configured to be adjacent to the second end of the gel strip chamber when
the chamber is covered.
3. The device of claim 2, wherein the cover includes a sample loading
reservoir.
4. The device of claim 2, wherein the cover includes a buffer introduction
reservoir.
5. The device of claim 2, wherein the cover includes a bottom surface
having a capillary break channel.
6. The device of claim 2, wherein the cover includes an oil reservoir that
communicates with the capillary break channel.
Description
FIELD OF THE INVENTION
The invention relates to devices and methods used in preparing gel strips
for electrophoresis and performing electrophoresis on gel strips.
BACKGROUND OF THE INVENTION
Two-dimensional electrophoresis is an effective way to analyze complex
mixtures of proteins. Typically, two-dimensional electrophoresis involves
separating the protein mixture by the intrinsic charge characteristics of
the proteins, i.e., their isoelectric points, in a first dimension by a
type of electrophoresis called isoelectric focusing, and then separating
the protein mixture in a second dimension by electrophoresis. In the
second-dimension electrophoresis, a gel strip containing the proteins
separated in the first dimension is incubated in a buffer appropriate for
the second-dimension electrophoresis, and applied to a second-dimension
vertical or horizontal slab gel so that the proteins can be
electrophoresed out of the first-dimension gel and into the second gel
under appropriate conditions to separate the proteins on the basis of
molecular mass.
The first-dimension electrophoresis, i.e., isoelectric focusing, is usually
performed on thin flat strips of polyacrylamide gel containing a
covalently immobilized pH gradient, i.e., IPG gel strips. The IPG gel
strips are commercially available in a dehydrated state and are rehydrated
in an appropriate buffer before use. Currently, each IPG gel strip is
rehydrated in a first gel carrier apparatus, and then handled and
transferred by the user to a second gel carrier apparatus for isoelectric
focusing (IEF) to separate the supplied proteins by isoelectric point.
A problem with the present processes and gel carrier apparatuses for IPG
gel strip preparation and isoelectric focusing is that the IPG gel strips
tend to be fragile, flimsy, and difficult to handle between the steps of
rehydration and first dimension electrophoresis, as is commonly practiced
at present time. Transferring the gel strip from a first gel carrier
apparatus for rehydration to a second gel carrier apparatus for
isoelectric focusing requires too much handling and hands-on-time for the
first-dimension electrophoresis.
SUMMARY OF THE INVENTION
To this end, a first aspect of the present invention involves a gel strip
carrier module for a gel strip that reduces the handling of the gel strip
and the hands-on-time during preparation of the gel strip for isoelectric
focusing by including a gel strip chamber that serves as both a
rehydration and focusing chamber.
A second aspect of the present invention involves a device for rehydrating
and performing electrophoresis on a gel strip having two ends and a gel
face. The device includes a holder having a gel strip chamber configured
to receive the gel strip and a rehydration buffer medium. First and second
electrodes are carried by the holder. The electrodes include internal
electrical contact points adapted to contact the gel face of the gel strip
near the ends of the gel strip and external electrically contact points
adapted to be electrical connected to a power supply for performing
electrophoresis on the gel strip.
A preferred embodiment of the above aspect of the invention includes a
number of features. A first feature is that the internal electrical
contact points are carried by the holder and are adapted to contact the
face of the gel strip with the gel strip oriented gel face down in the
chamber. A second feature is that the holder includes electrolytic gas
bubble escape vents comprised of a widened area in the chamber adjacent to
an internal electrical contact. A third feature is that the chamber may
include an optional sample introduction area between the internal
electrical contact points comprised of a widened area in the chamber
adjacent to a lateral edge of the gel strip. A fourth feature is that the
holder includes a floor and a wall, an inner surface of the wall and upper
surface of the floor define the gel strip chamber, the gel strip chamber
includes a first end and a second end, the internal electrical contact
point of the first electrode is carried by the floor adjacent to the first
end of the gel strip chamber, the internal electrical contact point of the
second electrode is carried by the floor adjacent to the second end of the
gel strip chamber. A sixth feature is that the gel carrier modules
includes a cover for the gel strip chamber. A seventh feature is that at
least one member is adapted to maintain the gel strip in contact with the
electrical contacts, and the at least one member extends from the cover
and includes a bottom surface and a height, whereby when the chamber is
covered, the distance between the floor and the bottom surface of the at
least one member is equal to the thickness of the gel strip when
rehydrated. An eighth feature is that the gel carrier module may include a
retaining mechanism adapted to retain the cover to the holder.
An alternative embodiment of the above aspect of the invention includes a
number of features. A first feature is that the internal electrical
contact points are carried by the cover and are adapted to contact the
face of the gel strip with the gel strip oriented face up in the chamber.
This embodiment of the invention may include a number of additional
features. A second feature is that the holder includes a floor and a wall,
an inner surface of the wall and upper surface of the floor define the gel
strip chamber, the gel strip chamber includes a first end and a second
end, the internal electrical contact point of the first electrode is
carried by the cover configured to be adjacent to the first end of the gel
strip chamber when the chamber is covered, the internal electrical contact
point of the second electrode is carried by the cover and configured to be
adjacent to the second end of the gel strip chamber when the chamber is
covered. A third feature is that the cover includes a bottom surface, and
the chamber includes a depth equal to the thickness of the gel strip when
rehydrated so that the bottom surface of the cover maintains the gel strip
in contact with the internal electrical contact points when the gel strip
is rehydrated. A fourth feature is that the cover includes a sample
loading reservoir. A fifth feature is that the cover includes a buffer
introduction reservoir. A sixth feature is that the cover includes a
bottom surface having a capillary break channel and an oil reservoir that
communicates with the capillary break channel.
A third aspect of the invention involves a device for rehydrating and
performing electrophoresis on a gel strip that includes a holder having a
gel strip chamber configured to receive the gel strip and a rehydration
buffer medium, and means for performing electrophoresis on the gel strip.
A preferred embodiment of the above aspect of the invention includes a
number of features. A first feature is that the device includes means for
venting electrolytic gas bubbles from the chamber. A second feature is
that device includes means for introducing a test sample to the gel strip.
A third feature is that the device includes means for retaining the cover
to the holder. A fourth feature is that the device includes means for
maintaining electrical contact between the gel strip and electrophoresis
means. A fifth feature is that the device includes means for accommodating
electroendosmotic flow in the gel strip during electrophoresis.
A fourth aspect of the invention involves a method of rehydrating and
performing electrophoresis on a gel strip having first and second ends and
a gel face. The method includes providing a device for rehydrating and
performing electrophoresis on the gel strip comprising a holder including
a gel strip chamber, the gel strip chamber configured to receive the gel
strip and a rehydration buffer medium, and first and second electrodes
having internal electrical contact points adapted to contact the gel face
within the gel strip chamber near the first and second ends of the gel
strip and external electrical contact points adapted to be electrically
connected to a power supply for performing electrophoresis on the gel
strip; providing a power supply; adding a rehydration buffer medium to the
gel strip chamber; placing the gel strip into the chamber; and performing
electrophoresis on the gel strip by applying voltage from the power supply
to the external electrical contact points.
A preferred embodiment of the above aspect of the invention may include a
number of features. A first feature is that the internal electrical
contact points are adapted to contact the face of the gel strip with the
gel strip oriented gel face down in the chamber, or the internal
electrical contact points are adapted to contact the gel face of the gel
strip with the gel strip oriented face up in the chamber. A second feature
is that the gel strip may be placed into the chamber gel face down after
the buffer medium is added to the rehydration chamber or gel face up
before the buffer medium is added to the rehydration chamber. A third
feature is that the method may include the step of providing an
experimental protein sample. A fourth feature is that the chamber may
include a sample introduction area between the internal electrical contact
points so that the step of providing an experimental protein sample
involves introducing the experimental protein sample in the sample
introduction area along a lateral edge of the gel strip after the gel
strip is lowered into the chamber, or alternatively, the experimental
protein sample may be included in the rehydration buffer medium. A fifth
feature is that the step of adding a rehydration buffer medium to the gel
strip chamber occurs before placing the gel strip into the chamber, and
the step of placing the gel strip into the chamber occurs before
performing electrophoresis on the gel strip. A sixth feature is that the
device includes a member adapted to maintain the gel strip in contact with
the internal electrical contact points during electrophoresis of the gel
strip. A seventh feature is that the device includes an electrolytic gas
bubble escape vent that allows electrolytic gases produced at the internal
electrical contact points during electrophoresis to escape. An eighth
feature is that the device includes a cover for the gel strip chamber.
Accordingly, the method further includes placing the cover on the gel
strip chamber.
Other features and advantages of the inventions are set forth in the
following detailed description and drawings, which are intended to
illustrate, but not limit, the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic view of an embodiment of a power application device,
shown with numerous other components of an electrophoresis unit;
FIG. 2 is a perspective view of an embodiment of a gel carrier module
resting on the power application device illustrated in FIG. 1;
FIG. 3 is a top plan view of the power application device illustrated in
FIG. 1;
FIG. 4 is a cross-sectional view of the gel carrier module illustrated in
FIG. 2;
FIG. 5 is an alternative embodiment of a power application device and a gel
carrier module;
FIG. 6 is a further embodiment of a power application device and a gel
carrier module;
FIG. 7 is a perspective view of an embodiment of an isoelectric focusing
unit;
FIG. 8 is an exploded perspective view of an embodiment of an IPG gel strip
carrier module and gel strip;
FIG. 9 is an exploded top plan view of the gel strip carrier module
illustrated in FIG. 8;
FIG. 10A is a cross sectional view of the gel strip carrier module
illustrated in FIG. 9, generally taken along line 10A--10A of FIG. 9, with
the gel strip shown in a dry state;
FIG. 10B is a cross sectional view of the gel strip carrier module
illustrated in FIG. 9, generally taken along line 10B--10B of FIG. 9, with
the gel strip shown in a rehydrated state;
FIG. 11 is a perspective sectional view of the gel carrier module
illustrated in FIG. 9, generally taken along line 11--11 of FIG. 9;
FIG. 12 is an exploded perspective view of an additional embodiment of a
gel strip carrier module;
FIGS. 13A-13F illustrate some of the steps of a process for rehydrating a
gel strip and performing isoelectric focusing on the gel strip;
FIG. 14 is an exploded perspective view of a further embodiment of a gel
strip carrier module;
FIG. 15 is a bottom perspective view of the cover of the gel strip carrier
module illustrated in FIG. 14;
FIG. 16A is a cross sectional view of a buffer reservoir opening and
electrode in the cover illustrated in FIG. 14;
FIG. 16B is a cross sectional view of a sample loading reservoir in the
cover illustrated in FIG. 14;
FIG. 16C is a cross sectional view of a pair of vents and capillary channel
in the cover illustrated in FIG. 14;
FIG. 17 is a partial cross sectional view of an end portion of the gel
strip carrier module illustrated in FIG. 14;
FIG. 18A is a cross sectional view of the gel strip carrier module
illustrated in FIG. 14 with a dry gel strip carried therein; and
FIG. 18B is a cross sectional view of the gel strip carrier module
illustrated in FIG. 14 with the gel strip shown in a rehydrated state;
DESCRIPTION OF THE PREFERRED EMBODIMENTS
An aspect of the present invention involves an improved device for applying
electrical power to a gel in an electrophoresis gel carrier module. With
reference to FIGS. 1-4, a preferred embodiment of a power application
device, which is indicated generally by the reference numeral 100, will
now be described in conjunction with a gel 102 that is carried by a gel
carrier module 104. The power application device 100 allows
electrophoresis gel carrier modules of various dimensions to be powered in
the same electrophoresis unit without adjustment of electrical contacts or
the use of conventional leads, plugs, and jacks, and provides a simpler
way to make high voltage contacts.
Following a description of the power supply device 100, an exemplary
embodiment of an isoelectric focusing (IEF) unit will be described in
conjunction with a gel strip carrier module that carries an immobilized pH
gradient (IPG) polyacrylamide gel strip. The IEF unit is a type of
electrophoresis unit used for first-dimension separation of complex
protein mixtures during two-dimensional electrophoresis, and incorporates
a power application device similar to the power application device 100.
With reference to FIG. 3, the power application device 100 includes a power
pad or platform 105 comprised of an electrically insulated support surface
106 that supports first and second electrically conductive regions or
contact areas, 108, 110, respectively.
In an embodiment, the support surface 106 is substantially rectangular and
is preferably made from an insulating material sold under the trademark
Kapton, manufactured by the E.I. duPont company. In alternative
embodiments, other insulating materials such as those used to make printed
circuit boards may be used, and/or the support surface may not be
rectangular.
The first electrically conductive region 108 serves as an anodic (+)
contact area and the second conductive region 110 serves as a cathodic (-)
contact area. Although the power pad 105 is described as having one set of
two conductive regions, it will be readily understood by those skilled in
the art that the power application device of the present invention may
include more than one set of conductive regions. Furthermore, although the
first conductive region is described as being anodic (+) and the second
conductive region is described as cathodic (-), the opposite may be true.
The conductive regions 108, 110 may be plated, imprinted or otherwise
adhered to the support surface 106. The conductive regions 108, 110 are
preferably made of copper and include a gold coating for purposes of low
resistivity and resistance to oxidation. In alternative embodiments, other
conductive materials may be used such as, for example, silver, gold,
copper, or other conductive materials or alloys.
The conductive regions 108, 110 may vary in size, shape, and orientation on
the support surface 106. In the preferred embodiment illustrated in FIG.
3, the first conductive region 108 is generally square and has a larger
area than the second conductive region 110, which is generally
rectangular. The flat, large-surface area construction of the conductive
regions 108, 110 allows electrophoresis gel carrier modules of various
dimensions to be powered in the same electrophoresis unit by simply
placing the gel carrier modules on the power pad 105.
It will be readily appreciated by those skilled in the art, that the size,
shape, and orientation of the conductive regions 108 may vary in order to
apply power to one or more gel modules having the same or different
dimensions.
In an alternative embodiment of the invention, the power pad 105 may be
constructed to allow for manual alteration of the configuration of the
conductive regions, allow replacement of the conductive regions, and/or
allow conductive regions to be added or taken from the power pad.
With reference to FIGS. 1 and 3, the conductive regions 108, 110, are
electrically coupled to a power supply 120 via conductive leads 112, 114
and power cables 116, 118. The power supply 120 is preferably integrated
with the power application device within an electrophoresis unit, and in
communication with a computer 132 for controlling the power supplied to
the conductive regions 108, 110. The first conductive region 108, i.e.,
anodic (+) contact area, is connected to an anodic (+) terminal of the
power supply 120 and the second conductive region 110, i.e., cathodic (-)
contact area, is connected to a cathodic (-) terminal of the power supply
120.
The power application device 100 preferably includes a temperature control
device or temperature control mechanism 122 integrated thereto and
efficient thermal contact therewith for controlling the temperature of the
gel during electrophoresis. One embodiment of a temperature control
mechanism 122 is illustrated in FIG. 1. The temperature control device 122
illustrated in FIG. 1 includes a heat distribution plate 124 with cooling
radiator fins 126 extending from its undersurface, and at least one
solid-state Peltier heating and cooling device 128 in efficient thermal
contact with the undersurface of the heat distribution plate and in
communication with a computer 132. The heat distribution plate 124 is in
efficient thermal contact with the power pad 105 through the insulated
support surface 106, which serves as a heat transfer means. A fan (not
shown) may be controlled by a computer 132 to draw air across radiator
fins 126 to maintain the efficiency of the Peltier heating and cooling
devices 128 when operating in cooling mode. Additionally, at least one
temperature sensor 130 is in efficient thermal contact with the support
surface 106, and is in communication with a computer 132. The temperature
sensor 130, computer 132, and Peltier heating and cooling device 128
function work together to control the temperature of the support surface
106, and in turn, the gel, during electrophoresis in response to a
selected temperature and electrophoresis voltage inputted into the
computer 132 by the user.
Cooling the gel during electrophoresis is important because it allows the
power application device 100 to be run at higher voltages without
overheating the gel. The electrophoretic separation of a protein mixture
test sample depends on the temperature of the gel during electrophoresis.
Temperature control allows for uniform separations and reproducible
separations. Many temperature control devices used with electrophoresis
units include bulky refrigeration units that circulate a refrigerant and
are located apart from the electrophoresis unit. These temperature control
devices occupy large amounts of bench space in a laboratory, and may not
control temperature as effectively as the present temperature control
device 122.
Although a single temperature control device 122 has been described in
conjunction with the power pad 105, the power pad 105 may have multiple
small temperature control devices to individually control the temperature
of multiple gels or multiple gel regions each at a selected different
temperature during electrophoresis.
With reference to FIGS. 1, 2, and 4, the gel carrier module 104, which is
constructed in accordance with an embodiment of the invention, will now be
described. The gel carrier module 104, when properly positioned on the
power pad 105, bridges or connects the first and second conductive regions
108, 110, respectively.
The gel carrier module 104 includes a substantially rectangular tray or
holder 133 having a base 134, two substantially parallel side walls 136,
and two substantially parallel end walls 138. The inside of the gel
carrier module 104 comprises a gel chamber that is defined by the inner
surfaces of the walls 136, 138 and an upper surface of the base 134. The
gel chamber has a configuration suitable for carrying the gel 102, any
applied mediums used during electrophoresis such as a buffer, test sample,
or the like. The gel may be made of a number of different substances such
as polyacrylamide, agarose, or the like.
The base 134 of the module 104 carries a first electrode or anodic (+)
electrode 140 having a first inner electrical contact point 142 and first
outer electrical contact point 144. The first inner electrical contact
point 142 is located in the gel chamber and is the point or area near one
end of the gel 102 where the gel directly contacts the first electrode 140
or indirectly contacts the first electrode through an appropriate
conductive solution. The first outer electrical contact point 144 is
located on the bottom surface of the base 134 and is the point where the
first electrode 140 contacts the first conductive region or anodic (+)
contact area 108.
The base 134 also carries a second electrode or cathodic electrode 146 near
an opposite end of the base 134 from the first electrode 140, and includes
a second inner electrical contact point 148 and a second outer electrical
contact point 150. The second inner electrical contact point 148 is
located in the gel chamber and is the point or area near one end of the
gel 102 where the gel directly contacts the second electrode 146 or
indirectly contacts the second electrode 146 through an appropriate
conductive solution. The second outer electrical contact point 150 is
located on the bottom surface of the base 134 and is the point where the
second electrode 146 contacts the second conductive region or cathodic (-)
contact area 110.
The conductive electrodes 140, 146 are preferably platinum wire or bands,
or the like, which span the width of the gel chamber. The conductive
electrodes 140, 146 extend beyond the side or end walls 136, 138, or
through the base 134 so that internal contact point 142, 148 and external
contact points 144, 150 exist.
Resting the gel carrier module 104 on the power platform 105 so that the
first and second outer electrical contact points 144, 150 of the first and
second electrodes 142, 146, respectively, electrically contact the first
and second conductive regions 108, 110, respectively, causes an electrical
connection to occur between the gel 102 and the power pad 105 suitable for
performing electrophoresis on the gel 102.
The gel carrier module 104 and/or power pad 105 may include appropriate
anodic (+) and/or cathodic (-) indicators for assisting the user in
properly orienting the gel 102 in the module 104 and/or properly orienting
the module 104 on the power pad 105 so that an appropriate electrical
connection is made between the conductive regions 108, 110 of the power
pad 105 and the ends of the gel 102.
Although not shown, the gel carrier module 104 may also include a cover or
lid that fits with and covers the top of the module. As will become better
understood below, electrical contact with the power pad can be made
through such a cover.
Although not shown, the gel carrier module 104 may also include means for
indicating dimensions, power requirements or limits, or other information
to a sensor embedded in the support surface 106. For example, the carrier
may include a magnetic or otherwise coded label that provides information
to a sensor in the support surface 106. The sensor would be in
communication with the computer to provide the computer with the coded
information. The computer may control power, temperature, data recording,
etc. based on the received information.
It is important for the support surface 106 and the opposing bottom of the
base 134 of the gel carrier module 104 to be sufficiently mutually flat or
non-interfering for the external contact points 144, 150 and conductive
regions 108, 110 so that secure electrical contact is made between the
contact points and conductive regions 108, 110. It is also important for
air gaps to be minimal between the support surface 106 and the bottom
surface of the gel module because the support surface serves as a heat
transfer means. It is also important that the degree of contact between
the external contact points 144, 150 and the respective conductive regions
108, 110 be sufficiently broad so that the requisite electrical current is
carried through the gel 102 without overheating or burning of the external
contact points 144, 150 or the conductive regions 108, 110.
As mentioned above, an important aspect of the power application device 100
of the present invention is that it can be used with one or more gel
carrier modules having a wide variety of structural and/or electrode
configurations, such as, but not limited to, the gel carrier modules
described herein. If the gel module has an anode (+) electrode capable of
making electrical contact with a gel it carries and the anode (+) contact
area 108 of the power application device 100, and a cathode (-) electrode
capable of making electrical contact with the gel it carries and the
cathode (-) contact area 110 of the power application device 100, the gel
carrier module will be appropriate for use with the power application
device of the present invention.
Although the power application device of the present invention has been
shown and described as having a generally horizontal orientation, the
power application device may have other orientations such as a generally
vertical orientation.
Accordingly, with reference to FIG. 5, a power application device 200 and
gel carrier module 202 in accordance with an additional embodiment of the
invention will now be described. The gel carrier module 202 carries a gel
204 for performing vertical electrophoresis on the gel 204. Similar to the
generally horizontal power application device 100 described above, the
power application device 200 includes a power pad or platform 205
comprised of an insulated support surface 206 and first and second
conductive regions 208, 210, respectively. The power pad 205 is generally
vertically oriented, supported by a frame (not shown), and includes a
rearwardly angled portion where the second conductive region 210 resides.
The angle of the rearward angled portion is greater than 90.degree. and
less than 180.degree., with reference to the face of most of the power pad
205. This rearwardly angled portion may be formed by bending the support
surface 206 between the conductive regions 208, 210, or by forming the
power platform 205 with two separate support surfaces, each carrying one
of the conductive regions 208, 210, and providing the support surface
carrying the second conductive region 210 at the aforementioned rearwardly
inclined angle.
Similar to the power application device 100 described above, the power
application device 200 illustrated in FIG. 5 preferably includes a
temperature control device 222 similar to the temperature control device
described above.
The gel carrier module 202 is effectively a folded version of the gel
carrier module 104 described above. The gel module 202 includes a front
wall 230 and a back wall 232. At a bottom portion of the walls 230, 232,
the gel module 202 includes a lower buffer chamber 234 defined by a lower
buffer chamber assembly 236. A first electrode 238 is carried by a lower
portion of the back wall 232, and includes an inner contact point 240 and
an outer contact point 242. When the gel carrier module 202 is lowered
onto the power pad 205, the outer contact point 242 makes electrical
contact with the first conductive region 208, i.e., anodic (+) contact
area. The first electrode 238 is electrically connected with a lower
surface 244 of the gel 204 through a conductive buffer 246 in the lower
buffer chamber 234.
At a top portion of the walls 230, 232, the gel module 202 includes an
upper buffer chamber 248 defined by an upper buffer chamber assembly 250.
The back wall 232 in the upper buffer chamber assembly 250 has a
rearwardly inclined angle similar to that of the power pad 205 so that the
gel carrier module 202 can be supported on the power pad 205 in this area.
A second electrode 252 is carried by the downwardly angled portion of the
back wall 232 and includes an inner contact point 254 and an outer contact
point 256. When the gel carrier module 202 is lowered onto the power pad
205, the outer contact point 256 makes electrical contact with the second
conductive region 210, i.e., cathodic (-) contact area. The second
electrode 252 is electrically connected with an upper surface 258 of the
gel 204 through a conductive buffer 260 which fills the upper buffer
chamber 248 and a sample well 262.
The electrically conductive gel 204 is contained between the vertical front
wall 230 and back wall 232 in a manner familiar to those experienced in
the art of vertical gel electrophoresis.
With reference to FIG. 6, a generally vertically oriented power application
device 300 constructed in accordance with an additional embodiment of the
invention is shown. The power application device 300 is similar to the
power application device 200 described above, except the upper buffer
chamber assembly of the embodiment of the gel carrier module and the upper
portion of the power pad shown in FIG. 6 are not rearwardly angled as in
the power application device 200 described in conjunction with FIG. 5.
The power application device 300 is used in conjunction with a gel carrier
module 302. The power application device 300 is carried by a frame 305
comprising a base 306 and a vertical support 308. The base 306 includes a
notch 310 in an upper surface of the base 306. A pivot clip 312 is
pivotally connect to a top part of the vertical support 308 through a pin
314.
The power application device 300 includes a flat, generally vertical power
pad or platform 315 that is carried by the vertical support 308. The power
pad 315 includes a vertical support surface 316, and first and second
conductive regions 318, 319.
The gel carrier module 302 includes a front wall 320 and a back wall 322.
At a lower portion of the walls 320, 322, the gel carrier module 302
includes a lower buffer chamber 324 defined by a lower buffer chamber
assembly 326. At a lower portion of the back wall 322, the back wall 322
carries a first electrode 328 having an inner contact point 330 and an
outer contact point 332. A wedge-like projection 333 extends from a lower
part of the lower buffer chamber assembly 326. When the gel carrier module
302 is properly positioned with the power application device 300, the
outer contact point 332 of the first electrode 328 contacts the first
conductive region 318 so that the first conductive region 318 is
electrically coupled to a bottom surface 334 of the gel 304 through a
conductive buffer 336 in the lower buffer chamber 324.
At an upper portion of the walls 320, 322, the gel carrier module 302
includes an upper buffer chamber 340 defined by an upper buffer chamber
assembly 342. At an upper portion of the back wall 322, the back wall 322
carries a second electrode 344 having an inner contact point 346 and an
outer contact point 348. When the gel carrier module 302 is properly
positioned with the power application device 300, the outer contact point
348 of the second electrode 344 contacts the second conductive region 319
so that the second conductive region 319 is electrically coupled to a top
surface 350 of the gel 304 through a conductive buffer 352 in the upper
buffer chamber 340.
The gel carrier module 302 is properly positioned with the power
application device 300 for vertical electrophoresis and held in this
position by first inserting the wedge-like projection 333 into the notch
310 of the base 306, and then retaining the top of the gel module 302 to
the top of the vertical support 308 by pivoting the pivot clip 312 over
the top of the back wall 322 of the gel carrier module 302.
With reference to FIG. 7, an isoelectric focusing unit 400 constructed in
accordance with a preferred embodiment of the invention will now be
described. An isoelectric focusing unit 400 is a type of electrophoresis
unit used for first-dimension separation of complex protein mixtures
during two-dimensional electrophoresis. The isoelectric focusing unit
illustrated in FIG. 7 includes a power application device 402, similar to
the power application device 100 described above, for performing
isoelectric focusing or separation, a type of electrophoresis, on a
immobilized pH gradient (IPG) polyacrylamide gel strip 403 (FIGS. 8-11)
carried by a gel carrier module 404 (FIGS. 8-11).
The isoelectric focusing unit 400 includes a generally rectangular housing
406 and a safety lid 408. The safety lid is pivotally connected to the
housing 406 at a top rear part of the housing 406 for opening and closing
the safety lid 408. A front part of the housing 406 includes an inclined
information display area 410. Information related to rehydration and
isoelectric focusing of the gel strip(s) is displayed in this area on a
screen 412. The display area 410 also includes a variety of input keys 414
for inputting information to be stored in the memory of a computer such as
the computer 132 described above with respect to FIG. 1. A top part of the
housing 406 supports the power application device 402.
The power application device 402 includes a power pad or platform 416
comprising an insulated support surface 418 and first and second
conductive regions, 420, 422, respectively. As mentioned above, the power
application device 402 is similar to the power application device 100
described above in conjunction with FIGS. 1-4, and for that reason, will
not be described in as much detail as above. The isoelectric focusing unit
400 can include a solid state Peltier temperature control device
integrated with the power application device 402, a computer, an
integrated power supply, and numerous power connections, all similar to
components described above with respect to FIG. 1. The solid state Peltier
temperature controller controls the temperature of the power pad 416 to a
predetermined range. For example in a preferred embodiment this
temperature range is 18-25.degree. C..+-.1.degree. C. In the preferred
embodiment, the power supply has 100 W of maximum power, a line voltage of
90 to 260 VAC, and delivers a voltage to the power platform 416 of 0-8000
V DC, and a current of 0 to 1.5 mA. The temperature of the gel strip(s)
and the voltage supplied by the power supply is controlled via the
computer in conjunction with computer software, and in response to user
input, which is described in more detail below.
Similar to the gel carrier modules described above, the gel strip carrier
module 404 rests on the power platform 416 for electrical connection and
temperature control of the IPG gel strip 403, and includes a first
electrode and a second electrode adapted to be electrically connected to
the power application device 402 at the first conductive region, i.e.,
anodic (+) contact area, and second conductive region, i.e., cathodic (-)
contact area, respectively. The power platform 416 is wide enough to
accommodate up to twelve gel strip carrier modules 404. The conductive
regions 420, 422 may be marked for proper placement of various lengths of
gel carrier modules, e.g., include outlines 423 or end lines where
different sized gel carrier modules should be positioned, and may be
marked to identify the conductive regions as being anodic (+) or cathodic
(-). Similarly, the gel carrier modules 404 may include some means to aid
in proper placement of the gel carrier module 404 on the power pad 416
such as by including a pointed tip at the end of the module 404 near the
anodic end of the gel carrier module, i.e. point is plus (+), or by anodic
(+) and/or cathodic (-) end indicators.
The power platform 416 serves as an electrical connector and a thermal
control system that links the gel carrier modules 404 to the programmable
8,000 V, 1.5 mA power supply and the Peltier solid state temperature
controller that maintains IPG gel strip temperature at 18-25.degree.
C..+-.1.degree.. Currently available electrophoresis units were limited by
a maximum rated voltage of usually .ltoreq.3500 V. The combination of high
voltage and efficient cooling in the present invention can reduce IPG
strip focusing time to as little as two to three hours, typically two to
four hours.
The integrated power supply and temperature control is programmable through
the input keys 414 in the display area 410, in conjunction with the
computer and software for the computer. The software allows for nine
isoelectric focusing programs or protocols, each at a selected temperature
with nine ramp or step voltage changes to be stored by the computer. Each
program may have a delayed start for isoelectric focusing so that
rehydration of the gel strips 403 can occur, allowing the user to load the
gel carrier modules 404 with sample in rehydration buffer in, for example,
the afternoon, then have isoelectric focusing start automatically during
the night. Because the isoelectric focusing requires only two to four
hours for the IPG gel strips 403, the first-dimension separation can be
completed, for example, overnight, by the start of the next work day.
Current first-dimension isoelectric systems can take as long as two days
for rehydration and isoelectric focusing. Each program may have a maximum
current limit and a maximum temperature controllable by the user.
The safety lid 408 covers the entire power pad 416 to protect a user from
the high voltage applied by the power pad 416 to the gel carrier module
404. For safety purposes, the isoelectric focusing unit 400 includes a
high voltage shut-off device that cuts the power being supplied to the
power pad 416 when the lid 408 is opened.
With reference to FIGS. 8-11, the gel strip carrier module 404, which is
constructed in accordance with a preferred embodiment of the invention,
will now be described. The gel carrier module 404 serves as a device for
both rehydration and isoelectric focusing, a type of electrophoresis, of
the immobilized pH gradient (IPG) strip 403. The length of the gel carrier
module 404 can vary to accommodate IPG gel strips of various lengths.
The preferred IPG strips 403 used in conjunction with the gel carrier
module 404 and IEF unit 400 are precast IPG polyacrylamide gel strips sold
under the trademark Immobiline DryStrip by Amersham Pharmacia Biotech of
Sweden. These strips are cast on a plastic backing 405, and are available
in a variety of lengths, pH ranges, and pH gradient shapes. These IPG
strips are preferred for inhibiting pH gradient distortion over time,
i.e., cathodic drift, physical distortion, or breaking during handling.
The gel carrier module 404, which is in accordance with another aspect of
the invention, includes an elongated, generally rectangular holder 430 and
cover 432. The holder 430 is comprised of a base 434 with a flat upper,
inner surface 436 and a flat lower, bottom surface 438, side walls 440
having an inner surface 442, and end walls 444 having inner surface 446.
The walls 440, 444 include a flat, upper surface 447. The inner surfaces
436, 442, and 446 define a substantially rectangular gel strip chamber 448
having a depth suitable to contain a rehydrated IPG gel strip and a
protective over-layer, if desired. The chamber 448 has a width and length
sufficient to accommodate the width and length dimensions of the gel strip
403 to be received in the chamber 448. A first electrode 450 having an
inner contact point 452, which is carried by the upper surface 436 of the
base, and an outer contact point 454 (FIG. 11), which is carried by the
bottom surface 438 of the base 434, is generally carried by the base 434
near an anodic (+) end of the gel carrier module 404, which, as mentioned
above, may be marked or shaped to reflect such to aid the user properly
orienting the gel strip 403 and/or gel module 404 during handling. A
second electrode 456 having an inner contact point 458, which is carried
by the upper surface 436 of the base 434, and an outer contact point (not
shown, but similar to outer contact point 458), which is carried by the
bottom surface 438 of the base 434, is generally carried by the base 434
near a cathodic (-) end of the gel carrier module 404, which like the
anodic (+) end, may be marked or shaped to reflect such to aid the user
properly orienting the strip 403 and/or the gel module 404 during
handling. The electrodes 450, 456 are preferably made of platinum bands
that penetrate through the bottom of the chamber 448 so that internal and
external contact points or areas are made. Electrodes making internal and
external contacts may be applied or constructed by means, such as, but not
by way of limitation, adhesives, electrodeposition, metalization, in-place
molding, or use of appropriately bent clips.
With reference to FIG. 12, an example of an alternative way to create
electrodes 450, 456 having internal and external contact points is by
forming the holder 430 with multiple notches 461 adjacent the electrodes
450, 456. This simplifies applying the electrodes 450, 456. After applying
the electrodes 450, 456, the notches are filled with a potting compound
462 to provide a water-tight seal.
In a further alternative embodiment of the gel strip carrier module, which
will be described in more detail below in conjunction with FIGS. 14-18,
the electrodes making internal and external contacts may be applied
through the cover of the gel carrier module.
With reference back to FIGS. 8-11, the chamber 448 of the holder 430 may be
widened near the electrodes 450, 456 to form gas bubble escape or vent
areas 464 for the gases created due to electrolysis of water at the
electrodes 450, 456 during electrophoresis. The gas bubble escape areas
464 help to prevent the gel strip 403 from being forced off of the
electrodes 450, 456 by the gas pressure created due to electrolysis of
water during electrophoresis.
The inner surface 442 of the side walls 440 may include one or more sample
introduction wells or areas 468 anywhere between the electrodes 450, 456.
Although the present invention has been described in conjunction with a
protein sample, it will be readily understood by those skilled in the art
how the present invention may be applied to other samples such as DNA,
RNA, amino acids, nucleic acids, and the like.
The cover 432 includes a flat bottom surface 470 that abuts a flat upper
surface 447 of the walls 440, 444 to preferably seal or cover the chamber
448 when in position on the holder reasonably tight so that minimal
evaporation occurs; however, the cover 432 can seal the chamber 448
liquid-tight or gas-tight. The cover may be clear so that the progress of
rehydration and isoelectric focusing can be monitored visually.
With reference especially to FIGS. 10A, 10B, and 11, at least one hold-down
or holding block 472 protrudes from the bottom surface 470 of the cover
432. When the cover 432 is properly positioned on the upper surface 447 of
the holder 430, the holding block 472 projects into the chamber 448,
leaving a distance between a bottom surface 474 of the holding block 474
and the upper surface 437 of the base 434 approximately equal to the fully
rehydrated thickness of the IPG gel strip 403 when rehydrated, with its
backing sheet 405 (FIG. 10B).
With reference specifically to FIG. 11, where the gel strip 403 crosses the
electrode 450, 456, the gel may be slightly compressed. It is at this
junction that electrolysis of water occurs, with the concomitant formation
of bubbles of oxygen gas (O.sub.2) and hydrogen gas (H.sub.2) at the first
electrode 450, i.e., anode (+), and the second electrode 456, i.e.,
cathode (-), respectively, during the process of electrophoresis. The
pressure maintained by the cover 432 and the hold-down blocks 474 inhibits
the gel strip 403 from being forced out of contact with the electrodes
450, 456 by the pressure of the gas bubbles as they evolve. The bubble
escape areas 464 allow the gases to escape the electrode contact area.
In general, the gel carrier module 404 is preferably manufactured by
injection molding the cover 432 of an acrylic material and molding the
holder 430 of an aluminum oxide ceramic, machining the appropriate
surfaces to make them flat, cut penetrations where the electrodes 450, 456
are to be provided, and then fire and braze the electrodes in place on the
base 434.
With reference to FIGS. 13A-13F, a method for rehydrating and performing
isoelectric focusing on the gel strip 403 using the isoelectric focusing
unit 400 will now be described. The method begins by removing a protective
film from the IPG gel strip 403. Next, a rehydration solution is added to
the gel strip chamber 448 of the gel carrier module 404 (FIG. 13A). The
rehydration solution preferably contains an experimental protein mixture
sample. The entire length of the IPG gel strip 403 is then set in the
rehydration solution by placing the IPG gel strip 403 in the chamber 448
of the gel module 404, gel-side facing down or gel face down (FIG. 13B).
During this step, the user gently lays the IPG gel strip 403 in the gel
carrier module 404, ensuring that the ends of the IPG gel strip are placed
over the electrodes 450, 456, and the entire gel surface is wetted between
the electrodes 450, 456. Next, if the protein sample was not included in
the rehydration solution discussed above, the protein mixture sample may
be applied in the sample introduction well 468 following the rehydration
step (FIG. 13C). IPG cover fluid, e.g., parafin oil, may then be carefully
applied along the length of the IPG gel strip to inhibit evaporation (FIG.
13D). Next, the cover 432 is seated or placed on the holder 430 (FIG.
13E). Finally, the gel carrier module 404 with gel strip 403 is properly
positioned on the power pad 416 (FIG. 13F) of the isoelectric focusing
unit 400, which applies a selected program of voltage steps to the
electrodes 450, 456 after the IPG gel strip 403 has had sufficient time to
rehydrate.
Results using the isoelectric focusing unit 400 in conjunction with the gel
carrier module 404 for extracts of E. coli on immobilized pH gradient gel
strips for two-dimensional electrophoresis were compared to results
achieved using conventional commercial equipment. The equipment used for
the second-dimension electrophoresis was the same for both.
The conventional equipment for the first-dimension electrophoresis
consisted of an electrophoresis unit with heat exchanger sold under the
name Multiphor II, IPG gel strip support module sold under the name
Immobiline Dry Strip gel kit, a thermostatted circulating bath sold under
the name MultiTemp III, a high voltage power supply sold under the name
EPS 3500 XL, and a reswelling tray sold under the name IPG Reswelling
Tray, all from Amersham Pharmacia Biotech.
After first-dimension electrophoresis, the proteins were electrophoresed
off of the strip and into a vertical slab electrophoresis unit sold under
the name SE 600 by Hoefer Pharmacia Biotech to perform second-dimension
electrophoresis. The equipment and procedures for second-dimension
electrophoresis was the same for both.
When isoelectric focusing of an additional sample of the same E. coli
extract was performed in the gel carrier module 404, and on the
isoelectric focusing unit 400, the results after vertical slab
electrophoresis were virtually indistinguishable, but the rehydration and
separation time was much less with the gel strip carrier module 404 and
isoelectric focusing unit 400 compared to the conventional equipment.
The gel strip carrier module 404 of the present invention reduces handling
of the IPG gel strips for the first-dimension electrophoresis by serving
as both a rehydration and focusing chamber for an IPG strip. The gel
carrier module 404 allows for the sample to be applied either through out
the entire gel or in a defined zone. When the sample is included in the
rehydration solution, the sample is loaded into the entire gel by
absorption during the rehydration step. Alternatively, the sample
introduction well 468 allows the sample to be applied in a defined zone
between the electrodes 450, 456. Since the rehydrated gel is in direct
contact with electrodes 450, 456 of the gel carrier module 400, the gel is
in position to run without further handling. Isoelectric focusing is
initiated by simply placing the gel carrier module 404, with IPG gel strip
403, rehydration solution, and sample, on the power platform or pad 416 of
the power application device 402, and selecting a protocol. The computer
of the isoelectric focusing unit applies power to the strip 403
automatically after a specified time for rehydration, without user
intervention.
With reference to FIGS. 14-18, a gel strip carrier module 500 constructed
in accordance with an additional embodiment of the invention will now be
described. The gel carrier module 500 includes an elongated generally
rectangular holder 502 and cover 504. The holder 502 includes one or more
rectangular notches 506 along a flat bottom surface 508 for receiving one
or more clips 510. The flat bottom surface 508 is necessary for making
substantial uniform contact with the power pad 416. When properly
positioned, each clip 510 serves as a clamping mechanism for retaining the
cover 504 and holder 502 together (FIGS. 18A, 18B). The clip 510 may be a
separate clip, as shown, a series of clips, or one or more clips
integrated into the holder 502 and/or cover 504.
The holder 502 has a shallow, flat-bottomed recess or gel strip chamber 512
along a flat top surface 514. The gel strip chamber 512 is defined by an
inner chamber wall 516 and a lower chamber surface 518. The inner wall 516
of the gel strip chamber 512 has a depth suitable to just contain the IPG
gel strip 403, gel side facing up, when rehydrated on its backing sheet
405.
With reference to FIG. 15, the cover 504 has a flat bottom surface 520
which seals against the flat top surface 514 of the holder 502. The cover
504 may include a buffer reservoir opening 522 near electrode lead holes
523 (FIG. 16A), a sample loading reservoir 524 (FIG. 16B), and a capillary
break channel 526 (FIG. 16C), which can optionally be filled with a light
oil through a pair of vents 528. The capillary break channel 526 can serve
as a capillary flow interrupter or can be filled with the light oil to
prevent both capillary flow and contact with atmospheric oxygen and carbon
dioxide. Without some sort of capillary break means, capillary creepage
tends to occur between the flat bottom surface 520 of the cover 504 and
the flat top surface 514 of the holder 502. This capillary creepage can
lead to leakage, corrosion, drying and other problems.
With reference to FIG. 17, an electrical assembly for making electrical
contact to the IPG gel strip 403 through the cover 504 will now be
described. At positions in the cover 504 corresponding to near the ends of
the gel strip chamber 512, electrode wires 530 are threaded through the
electrode lead holes 523, leaving an electrode segment 532 exposed for
contact with the gel or gel face of the IPG gel strip 403 when the cover
504 is properly positioned on the holder 502. An electrical circuit 533
from the electrical wires 530 continues to a metal leaf spring anchor
screw 534, which retains a conductive leaf spring 535, and to ball contact
536. The anchor screw 534 is received within an anchor screw opening 538.
The ball contact 536 is threadably attached to threaded fastener 540,
which is received with a fastener opening 542. Electrical power is applied
by the first and second conductive regions, 420, 422, of the power pad 416
to the IPG gel strip 403 through the ball contacts 536, which are located
at opposite ends of the gel carrier module 500. Accordingly, the
electrical circuit 533 functions as an electrode with the electrode
segment 532 as an internal contact point and the ball contact 536 as an
external contact point.
With reference to FIGS. 18A and 18B, the gel carrier module 500 is
assembled by placing a dry IPG gel strip in the gel strip chamber 512, gel
side facing up, positioning the cover 504 on the holder 502, and securing
the cover 504 to the holder 502 with the clips 510 as shown. At this point
the dry IPG gel strip will appear as shown in FIG. 18A. Upon addition of
an appropriate volume rehydration buffer, the dry IPG gel strip 403 swells
and makes contact with the electrode segment 532 (FIG. 18B). The buffer
may be pipetted through the reservoir openings 524 or pipetted onto the
gel face before applying the cover 504 to the holder 502. After allowing
an appropriate time for full rehydration to occur, an electrical field can
be applied via the power application device 402 to effect the
first-dimension separation.
Solvent can be introduced and/or removed, as may be required to accommodate
electroendosmotic flow during electrophoresis, through the buffer
reservoir openings 522.
It will be readily apparent to those skilled in the art how some of the
features described herein may be applied to other embodiments or aspects
of the invention.
Although this invention has been described in terms of certain preferred
embodiments, other embodiments apparent to those of ordinary skill in the
art are also within the scope of this invention. Accordingly, the scope of
the invention is intended to be defined only by the claims that follow.
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